Individual reflectance of solar radiation confers a thermoregulatory benefit to dimorphic males bees (Centris pallida) using distinct microclimates

Incoming solar radiation (wavelengths 290–2500 nm) significantly affects an organism’s thermal balance via radiative heat gain. Species adapted to different environments can differ in solar reflectance profiles. We hypothesized that conspecific individuals using thermally distinct microhabitats to engage in fitness-relevant behaviors would show intraspecific differences in reflectance: we predicted individuals that use hot microclimates (where radiative heat gain represents a greater thermoregulatory challenge) would be more reflective across the entire solar spectrum than those using cooler microclimates. Differences in near-infrared (NIR) reflectance (700–2500 nm) are strongly indicative of thermoregulatory adaptation as, unlike differences in visible reflectance (400–700 nm), they are not perceived by ecological or social partners. We tested these predictions in male Centris pallida (Hymenoptera: Apidae) bees from the Sonoran Desert. Male C. pallida use alternative reproductive tactics that are associated with distinct microclimates: Large-morph males, with paler visible coloration, behave in an extremely hot microclimate close to the ground, while small-morph males, with a dark brown dorsal coloration, frequently use cooler microclimates above the ground near vegetation. We found that large-morph males had higher reflectance of solar radiation (UV through NIR) resulting in lower solar absorption coefficients. This thermoregulatory adaptation was specific to the dorsal surface, and produced by differences in hair, not cuticle, characteristics. Our results showed that intraspecific variation in behavior, particular in relation to microclimate use, can generate unique thermal adaptations that changes the reflectance of shortwave radiation among individuals within the same population.


Introduction
Solar (shortwave) radiation plays a large role in an organism's thermoregulation via radiative heat gain, driving adaptive patterns of body coloration and reflectance [1,2]. Organisms may absorb solar energy to heat up, or transmit/reflect solar energy to avoid additional heat gain [3,  and plays a large role in allowing ectotherms to reach active temperatures at cool air temperatures [34]. Females may also be darker in coloration (like small-morph males) in order to increase solar radiative heat gain and maximize foraging performance in the cool, early-morning period. We hypothesized that microclimate usage drives morph-specific differences in UV-NIR reflectance. We predicted that large-morph males would have higher reflectance of solar radiation compared to small-morph males, as a thermoregulatory adaptation to reduce or increase shortwave radiative heat gain in their hotter or cooler microclimates, respectively. Because microhabitat differences are strongest in incident radiation from above, we predicted morph reflectance differences on the dorsal, but not ventral, body surfaces (see [8]). To better understand mechanisms of morph-specific reflectance we tested whether differences in reflectance were related to hair (branched setae) or cuticle characteristics; we measured solar reflectance of the dorsal surface of the thorax with, and without, hairs present. We predicted that hair density would correlate with higher reflectance across body regions, or morph/sex, and calculated hair density on the thorax and abdomen across morphs and females. Finally, we predicted that females would have similar reflectance profiles to small-morph males on the dorsal surface, and would not differ from males in ventral surface reflectance.

Specimen collection
We classified males as the 'large' male morph if we found them patrolling, digging, or fighting and they had the grey/white coloration (Fig 1, S1 Fig) and leg morphology (thick, bulging femurs on the hindmost pair of legs) distinctive to the large, behaviorally-inflexible morph [28][29][30][31][32][33][34][35][36][37][38][39]. We classified them as 'small' male morphs if we collected them hovering near vegetation (large-morph males never engage in hovering behavior [28]). We collected C. pallida males and females (n = 10/morph or sex) in late April and early May of 2018, or in late April and early May of 2019, either within 10 km of N33.464, W111.632 or N32.223, W11.008. Permits were obtained through the Desert Laboratory on Tumamoc Hill, or bees were collected non-commercially on public lands and no permits were required. We transported bees in a cooler on ice to a lab where we weighed them on an analytical balance (Metler Toledo AB54-S) to the nearest 0.1 mg.

Morphological measurements
We used digital calipers (Husky, 6 inch-3 mode model) to measure the body parts of the male and female bees used for reflectance spectrophotometry. We assumed the thorax and head to be a single cylinder for surface area calculations, with a diameter was equal to the widest point of the thorax and height equal to the length of the bee from the top of the head to the back of the thorax. We assumed the abdomen was a cylinder with a diameter equal to the width of the second tergite, and height equal to the length of the abdomen. For surface area calculations, we subtracted one flat, circular side (e.g., base) of both the thorax-head, and abdomen, cylinders, in order to account for the surface where the abdomen and thorax meet.

Reflectance spectrophotometry
We stored males of each morph and females dry, and away from light, following field collection. We captured total diffuse and spectral ultraviolet to near-infrared (290-2500 nm) percent reflectance (R) of the external surface using a Cary 5000 spectrophotometer equipped with an Agilent integrating sphere, deuterium arc (<350 nm) and tungsten-halogen (>350 nm) lamps, and R928 photomultiplier and PbS IR detectors. We limited measurements to a consistent diameter for all specimen using a custom aluminum aperture (2.18 mm diameter, 0.66 mm thickness), which we also used during the zero and baseline (Labsphere certified reflectance standard, Spectralon) measurements. We set integration time to 0.2 seconds, with data intervals of 1 nm. Baseline measurements varied across set up days, possibly due to minor contamination on the Spectralon surface, so we used the following formula to standardize measurements (R standardized,n ) across different set-ups: Where R n is the measured percent reflectance on Day n, B 100% is the Spectralon baseline (on Day 1 through Day n) and B 0% is the zero baseline (on Day 1 through Day n).
We measured R on the dorsal surface of the thorax (unshaved, and shaved of all hairs using a razor blade and the tips of #5 forceps), the dorsal surface of the abdomen on the first two terga, and the ventral surface of the abdomen on terga 2-3, for each specimen (n = 10 individuals of each morph/females for each area). We mounted specimen so that the thorax or abdomen was flat against the aperture, with measurements normal to the surface. Measurement error occurred between 799 and 800 nm due to the detector switching from the R928 to PbS IR (an artefact of sample orientation changing in relation to the new detector). To account for this, we took the difference in R between 799 and 800 for each individual and then added half of this value to all measurements � 799 and subtracted it from all values � 800. As C. pallida can lose hair over their lifetime, we only used individuals collected when the aggregation had just started, with low wing wear and no visible hair thinning on the thorax.
We assumed transmission through the bee was 0 (a standard assumption for insect bodies in the NIR [6,8]), and calculated average absorption (a n ) for a morph or sex at a wavelength 'n', using the following equation: where � R n is the average fraction of reflectance of all ten specimen at wavelength n.

Solar radiation calculations
Thermal flux due to solar radiation can be defined by summing direct beam (b), diffuse sky (d), and reflected (from substrates; r) radiation: where � a is the absorption coefficient of the bee (the fraction of beam, diffuse, or reflected radiation absorbed by the bee); A b , A d , and A r are the surface area of the bee exposed to beam, diffuse, or reflected radiation; and I b , I d , and I r is the total irradiance of the beam, diffuse, or reflected source. Here, we assumed A b = 0.25 A s (total surface area), and A d and A r = 0.5 A s (standard assumption for bees, and cylindrical objects, as in [36]). For A b and A d , we used the total thorax and abdomen surface areas separately for A s , in order to take advantage of the two distinct absorption coefficients we calculated for each region dorsally; for A r, we combined the two regions into a total surface area. Because only male morphs consistently differ in surface area, we did not perform thermal flux calculations for females; we did, however, calculate their absorption coefficients as described below.
We obtained the direct normal irradiance spectra in order to calculate � a b and � a r from the National Renewable Energy Laboratory (ASTM G173-03 Reference Air Mass 1.5 Spectra Derived from SMARTS v. 2.9.2 [37]). We calculated the direct beam absorption coefficient (a b ) for each individual on the dorsal surface of the abdomen and the thorax, as well as the shaved cuticle, between 290 and 2500 nm (λ min and λ max ) as: l min a n I n I b Where I n is the intensity of solar radiation at wavelength n and I b is the total direct beam irradiance between the min and max wavelengths.
We obtained the spectrum of radiation reflected from a light soil substrate (RS substrate ), at each wavelength (n), from the USGS Spectral Library (sample ID: splib07a record = 13249 [38]), in order to calculate the intensity of reflected radiation, I r , based on the intensity of direct beam solar radiation (I b ) at each wavelength (n).
We then calculated the average absorption coefficient ( � a r ) of reflected radiation, using each bee's ventral abdominal absorption (a rn ) coefficient at each wavelength (n): We also calculated a second measure of I r using an albedo measurement of light sand from the Sonoran Desert (I r = 0.245I b [39]), to compare with the light soil substrate spectrum obtained from the USGS spectral library. Unlike the USGS sample, this summed-reflectance value could not account for wavelength specific effects-but this value had the advantage of being from the location of interest.
We assumed � a d to be equal to � a b (as in [36]). We assumed I d was a 100 W/m 2 [40]. We estimated direct beam radiation on April 20 th , 2018 at 1000 hr using the following formulas from the solar radiation geometry literature [41][42][43]: With the following assumptions: S po = 1361 W/m 2 , a cd = 0.83 (clear day), and atmospheric pressure at the study site = standard air pressure (101.3 x 10 3 kPa). Altitude angle (ø) was calculated using the following equations, using the Julian date (J = 110), Latitude (32.2˚, 0.562 rad [λ]), time (t = 1000), and solar noon time (t 0 = 1200): solar zenith angle ðzÞ cos z ¼ ðsin l sin dÞ þ ðcos l cos d cos yÞ Finally, We divided Q solar by body mass (g) to determine solar radiative heat flux per unit of body mass.

Scanning electron microscopy
We shaved small patches of the second tergite of the abdomens of the specimen used for reflectance spectrophotometry (thoraxes were already shaved). We then mounted thoraxes and abdomens on metal stubs with electrically conductive tape and silver paint, before sputtercoating samples (Pt/Pd target, 80/20; Cressington 208 Hr Sputter Coater) for 40 s at 40 mA (approximately 8-10 nm deposition). We took three photos of the cuticular surface in three different places on the abdomen/thorax at 100X or 200X magnification using a Zeiss Supra 50VP (EHT set to 5 kV, high vacuum mode, SE2 detector). We used ImageJ to count the number of pores found at the base of the hairs within a standardized, circular region of 0.1 mm 2 area (centered on a pore) on each of the three photos for each individual, and averaged these three numbers to obtain hair density in terms of the number of hairs/mm 2 . On the abdomen, both unbranched and branched setae can be found, however the pores that at the base of these two setae types are quite distinct (Fig 2); we counted only the pore type that led to the branched setae, which was more common (in photo 2A, for instance, only 2.9% of pores are for unbranched setae).
We used GraphPad Prism 9.1.2 [44] and and R v. 4.1.3 [45] to analyze spectrophotometry, morphology, and hair density data. We used one-way ANOVAs with Tukey's MCT to compare body mass, and thorax and head or abdominal surface area across large/small males and females. We used a one-way ANOVA followed by Tukey's MCT for normally distributed data, or Kruskal-Wallis test with Dunn's MCT for data that were not normally distributed, to assess variation in hair density on the abdomen and thorax. We calculated mean percent reflectance in the UV (290-399), VIS (400-700) and NIR (split into "close NIR" [cNIR, 701-1400] and "far NIR" [fNIR, 1401-2500]) for each individual, and used a two-way RM ANOVA (with a Geisser-Greenhouse correction due to lack of sphericity in wavelength) to assess the affect of morph (e.g. large-morph male, small-morph male, and female), wavelength region (UV, VIS, cNIR, and fNIR), and individual differences in mean reflectance. When we found significant p-values for 'morph', we used a Tukey's MCT to assess variation across categorical morph assignments within each wavelength region (and adjusted p-values for multiple comparisons). To test for differences in mean reflectance in the UV, VIS, cNIR, and fNIR between the shaved and unshaved dorsal thorax surface, or the dorsal and ventral side of the abdomen, of large and small males, we used two-way RM ANOVAs with Geisser-Greenhouse corrections. When we found significant p-values for the effect of 'shaving' (thorax) or 'side' (abdomen), we used a Sidak's MCT to assess variation associated with that variable within each wavelength region (and adjusted p-values for multiple comparisons). To test for a specific thermal adaptation across morphs/sexes in the NIR on the dorsal surface of the thorax and abdomen, while controlling for correlations with VIS reflectance, we tested the morph/sex and mean VIS reflectance for each individual as fixed effect predictors of NIR reflectance in a linear model, using repeated single-term deletions based on AIC comparisons followed by Type 1 ANOVA p-value comparisons to determine the simplest, best-fit model. We used one-way ANOVAs followed by Tukey's MCTs were used to assess differences in absorption coefficients between morphs and females.

Reflectance of solar radiation
Morph/sex significantly affected mean reflectance on the dorsal abdomen and thorax surfaces (Fig 4, Table 3, S1 Table; Two-way RM ANOVA; dorsal-abdomen: F = 26.32, df = 2, Table 1. Differences between male morphs and females in their surface area and body mass (n = 10; One-way ANOVAs with Tukey's MCT). Different letters indicate statistically significant differences (p < 0.05), among morphs/sexes in that variable.

Variable
Morph/Sex Mean ± SD Range The only significant difference between morphs/sexes in the mean reflectance of the shaved dorsal thorax (e.g., cuticle-only) was between small males and females in the UV (Fig 4C, S2 Table; Tukey's: q = 5.70, df = 16.66, p = 0.0301). All differences in mean reflectance between large and small males on the thorax disappeared when the hairs were shaved (S2 Table; Tukey's: all p > 0.05). The presence of thorax hairs significantly increased mean reflectance across the entire UV-NIR spectrum for both small males and large males compared to just the cuticle (Table 4 & S3 Table; Two-way RM ANOVA with Sidak's MCT: all p < 0.05).

Table 2. Differences between male morphs and females in the density of hairs per mm 2 on their dorsal thorax (Kruskal-Wallis with Dunn's MCT) and abdominal surfaces (One-way ANOVA with Tukey's MCT).
Letters indicate significant differences (p < 0.05) across morphs/sex (n = 10) for that region. Asterisk indicates significant differences (p<0.05) between regions (abdomen vs. thorax) for that morph/sex (Kruskal-Wallis with Dunn's MCT).

PLOS ONE
The mean reflectance of the ventral abdominal surface did not differ across morphs/sexes (S2 Fig, Table 3; Two-way RM ANOVA: F = 0.65, df = 2, p = 0.53). The ventral surface of the abdomen of large males had lower mean reflectance across the entire UV-NIR spectrum compared to the dorsal surface (Table 4 & S3 Table; Two-way RM ANOVA with Sidak's MCT: all p < 0.05), however the mean reflectance of the dorsal and ventral surface did not differ in small males (F = 0.39, df = 1, p = 0.54).
Females and males did not differ in the direct beam absorption coefficients of their shaved cuticles (Fig 5A; One-way ANOVA, F = 2.78, df = 27, p = 0.08). There was also no difference in the absorption of reflected light by the ventral surfaces of male morphs and females (Figs 5B and 6C; One-way ANOVA, F = 1.21, df = 27, p = 0.31).
Direct beam and diffuse sky radiation was calculated to be 1,178.82 W/m 2 at 1000 h on April 20 th . Reflected radiative energy was higher when accounting for wavelength specific effects (329.85 W/m 2 ) as compared to when applying the same albedo for light sand (0.245) across all wavelengths (288.81 W/m 2 ); we used 329.85 W/m 2 for all further calculations.
Large-morph males absorbed a mean of 0.36 ± 0.02 W of thermal energy from reflected, direct, and diffuse radiative sources (1.28 ± 0.13 W/g of body mass), compared to 0.26 ± 0.02 Table 3 W for small-morph males (1.85 ± 0.21 W/g of body mass). If large-morph males had the same reflectance profiles as the averages for small-morph males, they would absorb 0.37 ± 0.03 W of thermal energy (or 1.40 ± 0.15 W/g of body mass, an 8-9% increase in W/g). Females absorbed a mean 0.28 ± 0.03 W of thermal energy, or 1.40 ± 0.11 W/g of body mass. For all three groups, hairs on the dorsal surface reduced thermal heat flux due to solar radiation; thermal flux for shaved large males was 0.41 ± 0.03 W (1.42 ± 0.17 W/g), for small males was 0.28 ± 0.02 W (1.94 ± 0.23 W/g), and for females was 0.31 ± 0.03 W (1.63 ± 0.12 W/g).

Discussion
Comparative studies of animal solar reflectance (or VIS coloration) often collect data at a macrogeographic scale, and demonstrate the effect of geographic gradients in climate variables (such as ambient temperature or solar radiation) on solar reflectance [2,4,11,16,19]. However, large differences in ambient temperature can occur within populations when individuals use different microclimates. The 1 cm above-ground microclimate where large male C. pallida engaged in their mating behaviors has air temperatures > 8˚C hotter than the 1 m aboveground hovering microclimate used by small males by 1100 am [32], demonstrating that there can be substantial differences in thermal selective pressure across distinct microclimates at the same site. The reduced solar absorption of large-morph males in both the VIS and NIR may be a unique thermal adaptation to this very hot microclimate, where they must behave for prolonged periods to successfully access mates (males are known to dig and fight for up to 19 minutes [28]). Morph-specific variation in solar reflectance on the dorsal surface went beyond the VIS, with consistent differences in reflectance in the NIR. Because morph reflectance differences were strongly correlated in the VIS and NIR, selection on visual characteristics (e.g., anti-predator adaptations) could indirectly affect NIR reflectance. However, the higher UV-NIR reflectance of large-morph males conferred a thermal benefit for this morph, which utilizes a hotter microclimate: the higher UV-NIR reflectance of large-morph males led to an 8-9% reduction in W/g of absorbed solar energy. We suggest that morph differences in solar reflectance may be, in part, a thermoregulatory adaptation to their distinct microclimates; reflectance differences beyond visible coloration suggests there may be non-visual selective factors partially or wholly driving reflectance differences.
Females had intermediate dorsal absorption coefficients compared to males: female thorax reflectance profiles were similar to small males, but lower compared to large males, and abdomen reflectance profiles were similar to large males, but higher compared to small males. There was no significant difference in the absorption of reflected irradiance on the ventral surface among morphs and sexes, supporting the hypothesis that differences in reflectance may be an adaptation that functions to increase radiative heat gain in cooler microclimates, and decrease it in hotter microclimates.
Differences in dorsal reflectance were entirely caused by the hairs; there was no significant difference in the shaved cuticular absorption coefficients of the male morphs or females. Large-morph males had higher hair densities on the thorax and abdominal surfaces compared to small-morph males; however, they had decreased hair density on the abdomen compared to females, despite similar reflectance profiles. This suggests that both hair density, as well as   [8]. The striated external surface structure of the ant's hairs contributes to total internal reflection; however, the lack of triangular shape, and the orientation of the hairs as perpendicular (as compared to parallel) to the cuticle, reduce the likelihood of this structural mechanism in the C. pallida case. Alternately, the hollow structure of the hair may play a role in thermal management, as is the case in polar bear fur [47] and the silver ant [8].
Temperature can vary both spatially and temporally at a given site: across the course of the morning, temperature increased from a low of 16.9˚C to a high of 44.5˚C between 0600 and 1100 hrs in the 1 cm microclimate, and from 16.1˚C to 38.9˚C in the 1 m microclimate [32]. In male Colias butterflies, intraspecific increases in radiative heat gain due to melanization allowed for longer flight periods at cooler air temperatures, increasing access to mating opportunities [48,49]. The mean body size of the C. pallida male population increases throughout the morning (both for males foraging, and males engaged in mating-relevant behaviors [50]), suggesting smaller males may use their darker coloration to increase radiative heat gain and their access to mates during cooler early morning periods when large males are not yet as prevalent in the population.
An additional implication of this work relates to unraveling the various factors that may lead to stabilizing selection for dimorphic body morphologies in alternative reproductive tactic systems. Generally, it has been hypothesized that bird predation may select against the largemorph male mating advantage in the C. pallida system, producing persistent size variation and two distinct morphs [34]. However, bird predation is variable across field sites [34] while size dimorphism is not. In addition, large-morph males are not behaviorally wary of predation events (which might be expected if predation was as a significant selective force); for example, large-morph males can be captured easily with just the fingertips and will continue to dig or fight when experimenter shadows pass over them [31,Barrett pers. obs.]. An alternate hypothesis, better supported by our data, relates the continued existence of two microclimate-specialized morphs to the thermoregulatory selective pressures of the available microclimates. Males of an intermediate size and UV-NIR reflectance profile would be disadvantaged due to higher metabolic rates (and thus higher metabolic heat production) when using small male mating strategies (in other bees with intraspecific body size variation, smaller individuals have increased power efficiency in flight without any additional metabolic cost [51]), while also disadvantaged due to their increased solar absorptivity when using the large male mating strategies. Individual morphological specialization in relation to body form and reflectance may potentially facilitate the stability of the male C. pallida alternative reproductive tactic system.
Our study demonstrates the importance of both VIS and NIR reflectance as intraspecifically traits conferring a thermal benefit to large-morph males in the hotter microclimate. Variation in reflectance of shortwave radiation, and other morphological adaptation to that benefit organisms facing thermal pressures (such as larger body sizes), may alter the necessity for physiological thermoregulatory differences-for example, large morph males that can avoid overheating in the sun due to their reduced relative solar heat load may not need higher thermal tolerances to survive (see [32]). The interplay between these morphological and physiological thermoregulatory benefits/strategies thus deserve greater attention. In addition, this variation suggests individuals within populations may find their fitness-relevant behaviors to be differently constrained by increasing global temperatures due to the effects of variation in shortwave radiative heat gain on their energy budget. If thermoregulatory selective pressures play a role in maintaining size or behavioral variation within species (such as alternative reproductive tactic systems), this suggests that climate change may have impacts that stretch beyond species-level diversity-affecting intraspecific morphological and behavioral diversity as well.
We demonstrate that individual differences, often overlooked, may be important for understanding the selective pressures acting on individuals to generate evolutionary adaptations to fine-scale ecological variation. Future studies should thus pay greater attention to the effects of morphological, physiological, or behavioral variation between conspecifics, which may have important consequences for understanding proximate and ultimate causes of ecological reflectance and coloration patterns.